The mechanism of action of a novel neuroprotective low molecular weight dextran sulphate: New platform therapy for neurodegenerative diseases like Amyotrophic Lateral Sclerosis.

Background: Acute and chronic neurodegenerative diseases represent an immense socioeconomic burden that drives the need for new disease modifying drugs. Common pathogenic mechanisms in these diseases are evident, suggesting that a platform neuroprotective therapy may offer effective treatments. Here we present evidence for the mode of pharmacological action of a novel neuroprotective low molecular weight dextran sulphate drug called ILB®. The working hypothesis was that ILB® acts via the activation of heparin-binding growth factors (HBGF). Methods: Pre-clinical and clinical (healthy people and patients with ALS) in vitro and in vivo studies evaluated the mode of action of ILB®. In vitro binding studies, functional assays and gene expression analyses were followed by the assessment of the drug effects in an animal model of severe traumatic brain injury (sTBI) using gene expression studies followed by functional analysis. Clinical data, to assess the hypothesized mode of action, are also presented from early phase clinical trials. Results: ILB® lengthened APTT time, acted as a competitive inhibitor for HGF-Glypican-3 binding, effected pulse release of heparin-binding growth factors (HBGF) into the circulation and modulated growth factor signaling pathways. Gene expression analysis demonstrated substantial similarities in the functional dysregulation induced by sTBI and various human neurodegenerative conditions and supported a cascading effect of ILB® on growth factor activation, followed by gene expression changes with profound beneficial effect on molecular and cellular functions affected by these diseases. The transcriptional signature of ILB® relevant to cell survival, inflammation, glutamate signaling, metabolism and synaptogenesis, are consistent with the activation of neuroprotective growth factors as was the ability of ILB® to elevate circulating levels of HGF in animal models and humans. Conclusion: ILB® releases, redistributes and modulates the bioactivity of HBGF that target disease compromised nervous tissues to initiate a cascade of transcriptional, metabolic and immunological effects that control glutamate toxicity, normalize tissue bioenergetics, and resolve inflammation to improve tissue function. This unique mechanism of action mobilizes and modulates naturally occurring tissue repair mechanisms to restore cellular homeostasis and function. The identified pharmacological impact of ILB® supports the potential to treat various acute and chronic neurodegenerative disease, including sTBI and ALS.

The application of nanoparticles in cancer immunotherapy: Targeting tumor microenvironment.

The tumor development and metastasis are closely related to the structure and function of the tumor microenvironment (TME). Recently, TME modulation strategies have attracted much attention in cancer immunotherapy. Despite the preliminary success of immunotherapeutic agents, their therapeutic effects have been restricted by the limited retention time of drugs in TME. Compared with traditional delivery systems, nanoparticles with unique physical properties and elaborate design can efficiently penetrate TME and specifically deliver to the major components in TME. In this review, we briefly introduce the substitutes of TME including dendritic cells, macrophages, fibroblasts, tumor vasculature, tumor-draining lymph nodes and hypoxic state, then review various nanoparticles targeting these components and their applications in tumor therapy. In addition, nanoparticles could be combined with other therapies, including chemotherapy, radiotherapy, and photodynamic therapy, however, the nanoplatform delivery system may not be effective in all types of tumors due to the heterogeneity of different tumors and individuals. The changes of TME at various stages during tumor development are required to be further elucidated so that more individualized nanoplatforms could be designed.

Perlecan domain I gradients establish stable biomimetic heparin binding growth factor gradients for cell migration in hydrogels.

Growth factor gradients orchestrate many biological processes including organogenesis, wound healing, cancer invasion, and metastasis. Heparin-binding growth factor (HBGF) gradients are established in living systems by proteoglycans including the extracellular matrix heparan sulfate proteoglycan, perlecan/HSPG2. Three potential HBGF-binding glycosaminoglycan attachment sites occur in N-terminal domain I of perlecan's five domains. Our overarching goal was to form stable, biomimetic non-covalently bound HBGF gradients surrounding cells encapsulated in hyaluronate-based hydrogels by first establishing perlecan domain I (PlnD1) gradients. A versatile multichannel gradient maker device (MGMD) was designed and 3D printed, then used to create desired gradients of microparticles in hydrogels. Next, we used the device to covalently incorporate gradients of PEGylated PlnD1 in hydrogels with high-low-high or high-medium-low concentrations across the hydrogel width. Fluorescently-labeled fibroblast growth factor-2 was delivered to hydrogels in phosphate-buffered saline and allowed to electrostatically bind to the covalently pre-incorporated PlnD1, producing stable non-covalent HBGF gradients. To test cell viability after flow through the MGMD, delicate primary human salivary stem/progenitor cells were encapsulated in gradient hydrogels where they showed high viability and continued to grow. Next, to test migratory behavior in response to HBGF gradients, two cell types, preosteoblastic MC3T3-E1 cell line and breast cancer cell line MDA-MB-231 were encapsulated in or adjacent to PlnD1-modified hydrogels. Both cell lines migrated toward HBGFs bound to PlnD1. We conclude that establishing covalently-bound PlnD1 gradients in hydrogels provides a new means to establish physiologically-relevant gradients of HBGFs that are useful for a variety of applications in tissue engineering and cancer biology. STATEMENT OF SIGNIFICANCE: Gradients of heparin binding growth factors (HBGFs) direct cell behavior in living systems. HBGFs bind electrostatically to gradients of HS proteoglycans in the extracellular matrix creating HBGF gradients. We recreated HBGF gradients in physiological hyaluronate-based hydrogels using a 3D-printed multichannel gradient maker device (MGMD) that created gradients of HS proteoglycan-derived perlecan/HSPG2 domain I. We demonstrated the ability of a variety of cells, including primary salivary stem/progenitor cells, pre-osteoblastic cells and an invasive breast cancer cell line, to be co-encapsulated in gradient hydrogels by flowing them together through the MGMD. The versatile device and the ability to create HBGF gradients in hydrogels for a variety of applications is innovative and of broad utility in both cancer biology and tissue engineering applications.

Tethered growth factors on biocompatible scaffolds improve stemness of cultured rat and human neural stem cells and growth of oligodendrocyte progenitors.

Currently, there is no widely accepted technique to efficiently and reproducibly grow stem and progenitor cells in vitro. Stem cells require contact with extracellular matrices as well as signals from growth factors to proliferate and to retain their stemness. We have shown a novel tissue culture platform (StemTrix cultureware) that transforms standard tissue culture plasticware into a multi-functional chitosan-based scaffold that supports the expansion of neural stem cells. The StemTrix scaffold is comprised of chitosan with immobilized heparin which in turn tethers heparin-binding growth factors. The scaffold is also coated with an adhesive ECM protein. Here we demonstrate that fibronectin or the RGD peptide contained in fibronectin are equally effective in promoting the adhesion, viability and growth of rat and human neural stem cells. When FGF-2 and heparin-binding EGF are tethered to the StemTrix cultureware neural stem cells grow ∼3 times faster and remain in a more primitive state as determined by both Western Blot and gene expression analyses. Another important feature of this new culture platform is that the NSCs remain in a primitive and proliferative state for 4days without refreshing the culture medium or providing new growth factors, which represents a 20-fold extension of FGF-2's biological activity vs when it is freely soluble in the medium. To test the utility of this scaffold for propagating other types of stem cells and progenitors we tethered platelet-derived growth factor (PDGF) and FGF-2 alone and in combination to the scaffold and tested the efficacy of this platform to maintain primary oligodendrocyte progenitors or the CG-4 cell line in a primitive state. Oligodendrocyte progenitors plated onto this multifunctional film proliferated for at least 3days without providing soluble growth factors while inhibiting the expression of the differentiation marker myelin-basic protein. Oligodendrocyte progenitors proliferated 3 times more rapidly than cells maintained on fibronectin-coated culture substrates in culture medium supplemented with soluble FGF-2 and PDGF. Finally, we show that StemTrix cultureware can be produced using clinical grade components, providing users with a fully defined platform suitable for clinical use that maintains stem cells or progenitors in a more uniform and primitive state.

Combined delivery of FGF-2, TGF-ß1, and adipose-derived stem cells from an engineered periosteum to a critical-sized mouse femur defect.

Critical-sized long bone defects suffer from complications including impaired healing and non-union due to substandard healing and integration of devitalized bone allograft. Removal of the periosteum contributes to the limited healing of bone allografts. Restoring a periosteum on bone allografts may provide improved allograft healing and integration. This article reports a polysaccharide-based tissue engineered periosteum that delivers basic fibroblast growth factor (FGF-2), transforming growth factor-ß1 (TGF-ß1), and adipose-derived mesenchymal stem cells (ASCs) to a critical-sized mouse femur defect. The tissue engineered periosteum was evaluated for improving bone allograft healing and incorporation by locally delivering FGF-2, TGF-ß1, and supporting ASCs transplantation. ASCs were successfully delivered and longitudinally tracked at the defect site for at least 7 days post operation with delivered FGF-2 and TGF-ß1 showing a mitogenic effect on the ASCs. At 6 weeks post implantation, data showed a non-significant increase in normalized bone callus volume. However, union ratio analysis showed a significant inhibition in allograft incorporation, confirmed by histological analysis, due to loosening of the nanofiber coating from the allograft surface. Ultimately, this investigation shows our tissue engineered periosteum can deliver FGF-2, TGF-ß1, and ASCs to a mouse critical-sized femur defect and further optimization may yield improved bone allograft healing. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 900-911, 2017.